Wang et al. Chinese Journal of Cancer (2015) 34:8 DOI 10.1186/s40880-015-0012-z
ORIGINAL ARTICLE
Open Access
Selective killing of K-ras–transformed pancreatic cancer cells by targeting NAD(P)H oxidase Peng Wang1,2†, Yi-Chen Sun1†, Wen-Hua Lu1, Peng Huang1,3* and Yumin Hu1*
Abstract Introduction: Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and plays a critical role in the pathogenesis of this malignancy. Increase of reactive oxygen species (ROS) has also been observed in a wide spectrum of cancers. This study aimed to investigate the mechanistic association between K-ras–induced transformation and increased ROS stress and its therapeutic implications in pancreatic cancer. Methods: ROS level, NADPH oxidase (NOX) activity and expression, and cell invasion were examined in human pancreatic duct epithelial E6E7 cells transfected with K-rasG12V compared with parental E6E7 cells. The cytotoxic effect and antitumor effect of capsaicin, a NOX inhibitor, were also tested in vitro and in vivo. Results: K-ras transfection caused activation of the membrane-associated redox enzyme NOX and elevated ROS generation through the phosphatidylinositol 3′-kinase (PI3K) pathway. Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras–transformed cells compared with parental E6E7 cells. Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras–transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells. In vivo, capsaicin exhibited antitumor activity against pancreatic cancer and showed oxidative damage to the xenograft tumor cells. Conclusions: K-ras oncogenic signaling causes increased ROS stress through NOX, and abnormal ROS stress can selectively kill tumor cells by using NOX inhibitors. Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras–transformed cells through a redox-mediated mechanism. Keywords: K-ras, Pancreatic cancer, Reactive oxygen species, NADPH oxidase, Capsaicin
Background Oncogenic mutations of the KRAS gene are present in >90% of pancreatic ductal carcinoma [1], which is an aggressive and deadly cancer [2]. Since pancreatic ductal carcinoma is unusually resistant to chemotherapy and radiation therapy and little progress has been achieved in the treatment of pancreatic cancer, surgical resection remains to be the only potentially curative therapy. The potential discoveries of pancreatic cancer therapeutics rely on advances in our understanding of the biology of the disease. Genetic lesions, including mutations of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), * Correspondence: [email protected]; [email protected] † Equal contributors 1 Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong 510060, P.R. China 3 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Full list of author information is available at the end of the article
cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein 53 (TP53), breast cancer 2 (BRCA2), and mothers against decapentaplegic homolog 4 (SMD4/DPC4), have been thought to contribute to the evolution of pancreatic adenocarcinoma [3]. Activating KRAS mutations are found in more than 90% of pancreatic adenocarcinomas and are highly associated with disease progression due to the activation of several effector pathways that induce cell proliferation, survival, invasion, and metabolic alterations [3-5]. Given the almost ubiquitous occurrence of K-ras mutations and its critical role in the development of pancreatic cancer, the ideal therapeutic strategy would be the direct blocking of KRAS oncogenic signaling. However, an effective small-molecule inhibitor of KRAS has yet to be identified [6]. Whereas the major effector proteins, such as Raf kinase, phosphatidylinositol 3′-kinase (PI3K), and RalGDS, play vital roles in Ras transformation, accumulating
© 2015 Wang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. Chinese Journal of Cancer (2015) 34:8
evidence has shown that reactive oxygen species (ROS) may serve as a messenger of Ras in signaling transduction pathways and that moderate increases in ROS levels may promote cell proliferation and contribute to cancer development [7,8]. Therefore, ROS appear to be an important downstream effector of Ras transformation in cancer cells. The role of the membrane-associated NADPH oxidase (NOX) in non-mitochondrial formation of ROS has been observed in various studies [9-11]. The activation or up-regulation of NOX has also been shown to play an important role in maintaining the cancer phenotype through stimulating the production of ROS [12-14]. The previous findings prompted us to investigate whether Kras oncogenic signaling increases ROS levels through the activation of NOX and whether modulators of NOX could provide a potential therapeutic opportunity for pancreatic cancer through a redox-mediated mechanism. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a natural compound, is a pungent ingredient found in a variety of red peppers and has been shown to inhibit cell surface NOX activity [15,16]. In the current study, we aimed to determine the mechanistic role of NOX in mediating ROS generation induced by K-ras oncogenic signaling. We compared ROS production as well as the expressions and activities of NOX in parental human pancreatic duct epithelial E6E7 cells and K-ras–transformed E6E7 cells, which have previously been shown to be highly tumorigenic [17]. We also examined the effect of capsaicin on parental and K-ras–transformed E6E7 cells. Importantly, the role of NOX-derived ROS generation in capsaicin-induced cytotoxicity was tested in K-ras–transformed E6E7 cells in comparison with parental E6E7 cells.
Methods Antibodies and reagents
The following antibodies were used for immunoblotting analysis using standard Western blotting procedures: superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), p22phox, and p-p40phox were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin, tublin, diphenyleneiodonium chloride (DPI), and capsaicin were purchased from Sigma-Aldrich, St. Louis, MS, USA. Cell culture
The parental E6E7 cell line and K-ras–transformed cell line, which had been established by transfecting the immortalized human pancreatic duct epithelial E6E7 cell line with K-rasG12V, were kindly provided by Dr. Paul Chiao from The University of Texas, MD Anderson Cancer Center and were cultured as reported previously [17]. Primary pancreatic cancer cell lines, including AsPC-1, Capan-1, and Panc-1, were obtained from American Type Culture Collection (ATCC) and cultured
Page 2 of 11
in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS). Quantitative real-time Polymerase Chain Reaction (PCR) analysis
The sequences for the genes to be measured are as follows: 5′-GGAGTTTCAAGATGCGTGGAAACTA-3′ (sense) and 5′-GCCAGACTCAGAGTTGGAGATGCT-3′ (antisense) for NOX2, 5′-CAAGCCGTGACCAAGGA CACCTG-3′ (sense) and 5′-CACACAGGACATCCACC GTGTC-3′ (antisense) for NOXA1. Real-time PCR analysis was performed by using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Otsu, Shiga, Japan) and Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). MTT assay
Cell growth was determined using MTT reagent in 96-well plates. After incubation, 20 μL MTT reagent was added to each well and incubated for an additional 4 hours and then the supernatant was removed. The cell pellets were dissolved in 200 μL DMSO. Absorbance was determined using a MultiSkan plate reader (Thermo, Helsinki, USA) at a wavelength of 570 nm. Colony formation assay
Cells were seeded in six-well plates and cultured for about 2 weeks. Colonies were fixed with methanol for 10 minutes and stained with crystal violet solution (Beyotime, Jiangsu, China) for 30 minutes. All the experiment was repeated 3 times. NOX activity
Cells were suspended in lysis buffer containing 20 mmol/ L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA,1 mmol/L EGTA, 100 mmol/L sucrose, and a cocktail of protease inhibitors. After homogenization, the samples were centrifuged at 800 g at 4°C for 5 minutes to pellet unbroken cells and nuclei. The supernatants were centrifuged at 100,000 g for 30 minutes to separate the membrane fraction (pellet) and the cytosolic fraction (supernatant). NOX activity was measured by lucigeninderived chemiluminescence, with 100 μmol/L NADPH or NADH as substrate, 50 μmol/L lucigenin, and 25 μg of cell membrane proteins. Chemiluminescence was measured using a luminometer (Turner Designs, Sunnyvale, CA, USA) for 1 minute. The signal was normalized and expressed as arbitrary light units per microgram protein per minute. Rac activity
The Rac activity assay was performed using the Rac-GEF (guanine-nucleotide exchange factors) Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, cells were washed in cold PBS, lysed in 1× Assay/Lysis Buffer, and centrifuged
Wang et al. Chinese Journal of Cancer (2015) 34:8
Page 3 of 11
for 10 minutes at 14,000 g at 4°C. Aliquots from the supernatant were used for determining protein concentration. The supernatant was incubated with nucleotide-free Rac1 G15A agarose beads to pull down the active form of RacGEFs. The beads were washed 3 times with 1× Assay/Lysis Buffer, and the bound proteins were eluted. The active Rac proteins were detected by Western blotting using an antiRac-GEF antibody (Tiam1).
of 0.9% sodium chloride solution by intraperitoneal injection. Five weeks after inoculation, all mice were euthanized and the tumor weights were measured. Animal experiments were approved by Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center and performed under the guidelines of the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996).
Invasion assay
Immunohistochemistry and TUNEL assay
Invasion assays were performed with BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). Pre-coated filter Matrigel inserts were rehydrated with 0.5 mL of PBS for 2 hours in humidified tissue culture incubator at 37°C in 5% CO2 atmosphere. After rehydration, PBS was removed. Then, 1 × 105 parental or K-ras–transformed E6E7 cells and Capan-1, AsPC-1, Panc-1 cells in 0.5 mL of supplement-free medium with or without 10 μmol/L capsaicin were seeded onto the upper part of each chamber insert, and the 24-well plates were filled with 0.5 mL of their culture medium. Following incubation for 16 hours, noninvaded cells on the upper surface of the insert were wiped off with a cotton swab, and the cells that had migrated onto the lower surface of the filter, were fixed and stained with the Hema 3 Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA) containing a fixative and 2 stain solutions. The inserts were air dried and photographed. Invasiveness was determined by counting cells in 3 microscopic fields (×100) per well, and the extent of invasion was expressed as an average number of cells per microscopic field.
Representative tumor tissues were sectioned and embedded in paraffin. The slides were then incubated with the primary antibody (mouse anti–8-oxoguanine monoclonal antibody, Abcam, Cambridge, UK) at 1:200 dilution overnight in a humidified chamber at 4°C. The slides were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Envision Detection Kit, Dako, Glostrup, Denmark) at 37°C for 30 minutes. Finally, the samples were stained with 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin and eosin (HE). Tumor cell death induced by capsaicin was detected by TUNEL assay with the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions. Statistical analysis
Statistical significant differences were evaluated by using Student’s t test (Prism GraphPad, San Diego, CA, USA). The Kolmogorov-Smirnov test (Cell Quest Pro software, Becton-Dickinson, San Jose, CA, USA) was used to evaluate the significant difference between control and treatment groups in flow cytometry analysis. A P value of
ORIGINAL ARTICLE
Open Access
Selective killing of K-ras–transformed pancreatic cancer cells by targeting NAD(P)H oxidase Peng Wang1,2†, Yi-Chen Sun1†, Wen-Hua Lu1, Peng Huang1,3* and Yumin Hu1*
Abstract Introduction: Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and plays a critical role in the pathogenesis of this malignancy. Increase of reactive oxygen species (ROS) has also been observed in a wide spectrum of cancers. This study aimed to investigate the mechanistic association between K-ras–induced transformation and increased ROS stress and its therapeutic implications in pancreatic cancer. Methods: ROS level, NADPH oxidase (NOX) activity and expression, and cell invasion were examined in human pancreatic duct epithelial E6E7 cells transfected with K-rasG12V compared with parental E6E7 cells. The cytotoxic effect and antitumor effect of capsaicin, a NOX inhibitor, were also tested in vitro and in vivo. Results: K-ras transfection caused activation of the membrane-associated redox enzyme NOX and elevated ROS generation through the phosphatidylinositol 3′-kinase (PI3K) pathway. Importantly, capsaicin preferentially inhibited the enzyme activity of NOX and induced severe ROS accumulation in K-ras–transformed cells compared with parental E6E7 cells. Furthermore, capsaicin effectively inhibited cell proliferation, prevented invasiveness of K-ras–transformed pancreatic cancer cells, and caused minimum toxicity to parental E6E7 cells. In vivo, capsaicin exhibited antitumor activity against pancreatic cancer and showed oxidative damage to the xenograft tumor cells. Conclusions: K-ras oncogenic signaling causes increased ROS stress through NOX, and abnormal ROS stress can selectively kill tumor cells by using NOX inhibitors. Our study provides a basis for developing a novel therapeutic strategy to effectively kill K-ras–transformed cells through a redox-mediated mechanism. Keywords: K-ras, Pancreatic cancer, Reactive oxygen species, NADPH oxidase, Capsaicin
Background Oncogenic mutations of the KRAS gene are present in >90% of pancreatic ductal carcinoma [1], which is an aggressive and deadly cancer [2]. Since pancreatic ductal carcinoma is unusually resistant to chemotherapy and radiation therapy and little progress has been achieved in the treatment of pancreatic cancer, surgical resection remains to be the only potentially curative therapy. The potential discoveries of pancreatic cancer therapeutics rely on advances in our understanding of the biology of the disease. Genetic lesions, including mutations of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), * Correspondence: [email protected]; [email protected] † Equal contributors 1 Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong 510060, P.R. China 3 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Full list of author information is available at the end of the article
cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein 53 (TP53), breast cancer 2 (BRCA2), and mothers against decapentaplegic homolog 4 (SMD4/DPC4), have been thought to contribute to the evolution of pancreatic adenocarcinoma [3]. Activating KRAS mutations are found in more than 90% of pancreatic adenocarcinomas and are highly associated with disease progression due to the activation of several effector pathways that induce cell proliferation, survival, invasion, and metabolic alterations [3-5]. Given the almost ubiquitous occurrence of K-ras mutations and its critical role in the development of pancreatic cancer, the ideal therapeutic strategy would be the direct blocking of KRAS oncogenic signaling. However, an effective small-molecule inhibitor of KRAS has yet to be identified [6]. Whereas the major effector proteins, such as Raf kinase, phosphatidylinositol 3′-kinase (PI3K), and RalGDS, play vital roles in Ras transformation, accumulating
© 2015 Wang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wang et al. Chinese Journal of Cancer (2015) 34:8
evidence has shown that reactive oxygen species (ROS) may serve as a messenger of Ras in signaling transduction pathways and that moderate increases in ROS levels may promote cell proliferation and contribute to cancer development [7,8]. Therefore, ROS appear to be an important downstream effector of Ras transformation in cancer cells. The role of the membrane-associated NADPH oxidase (NOX) in non-mitochondrial formation of ROS has been observed in various studies [9-11]. The activation or up-regulation of NOX has also been shown to play an important role in maintaining the cancer phenotype through stimulating the production of ROS [12-14]. The previous findings prompted us to investigate whether Kras oncogenic signaling increases ROS levels through the activation of NOX and whether modulators of NOX could provide a potential therapeutic opportunity for pancreatic cancer through a redox-mediated mechanism. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a natural compound, is a pungent ingredient found in a variety of red peppers and has been shown to inhibit cell surface NOX activity [15,16]. In the current study, we aimed to determine the mechanistic role of NOX in mediating ROS generation induced by K-ras oncogenic signaling. We compared ROS production as well as the expressions and activities of NOX in parental human pancreatic duct epithelial E6E7 cells and K-ras–transformed E6E7 cells, which have previously been shown to be highly tumorigenic [17]. We also examined the effect of capsaicin on parental and K-ras–transformed E6E7 cells. Importantly, the role of NOX-derived ROS generation in capsaicin-induced cytotoxicity was tested in K-ras–transformed E6E7 cells in comparison with parental E6E7 cells.
Methods Antibodies and reagents
The following antibodies were used for immunoblotting analysis using standard Western blotting procedures: superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), p22phox, and p-p40phox were purchased from Santa Cruz Biotechnology, Dallas, TX, USA; β-actin, tublin, diphenyleneiodonium chloride (DPI), and capsaicin were purchased from Sigma-Aldrich, St. Louis, MS, USA. Cell culture
The parental E6E7 cell line and K-ras–transformed cell line, which had been established by transfecting the immortalized human pancreatic duct epithelial E6E7 cell line with K-rasG12V, were kindly provided by Dr. Paul Chiao from The University of Texas, MD Anderson Cancer Center and were cultured as reported previously [17]. Primary pancreatic cancer cell lines, including AsPC-1, Capan-1, and Panc-1, were obtained from American Type Culture Collection (ATCC) and cultured
Page 2 of 11
in Dulbecco’s Modified Eagle’s medium (DMEM) with 10% Fetal bovine serum (FBS). Quantitative real-time Polymerase Chain Reaction (PCR) analysis
The sequences for the genes to be measured are as follows: 5′-GGAGTTTCAAGATGCGTGGAAACTA-3′ (sense) and 5′-GCCAGACTCAGAGTTGGAGATGCT-3′ (antisense) for NOX2, 5′-CAAGCCGTGACCAAGGA CACCTG-3′ (sense) and 5′-CACACAGGACATCCACC GTGTC-3′ (antisense) for NOXA1. Real-time PCR analysis was performed by using the SYBR Premix Ex Taq II kit (TaKaRa Bio, Otsu, Shiga, Japan) and Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA). MTT assay
Cell growth was determined using MTT reagent in 96-well plates. After incubation, 20 μL MTT reagent was added to each well and incubated for an additional 4 hours and then the supernatant was removed. The cell pellets were dissolved in 200 μL DMSO. Absorbance was determined using a MultiSkan plate reader (Thermo, Helsinki, USA) at a wavelength of 570 nm. Colony formation assay
Cells were seeded in six-well plates and cultured for about 2 weeks. Colonies were fixed with methanol for 10 minutes and stained with crystal violet solution (Beyotime, Jiangsu, China) for 30 minutes. All the experiment was repeated 3 times. NOX activity
Cells were suspended in lysis buffer containing 20 mmol/ L HEPES, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA,1 mmol/L EGTA, 100 mmol/L sucrose, and a cocktail of protease inhibitors. After homogenization, the samples were centrifuged at 800 g at 4°C for 5 minutes to pellet unbroken cells and nuclei. The supernatants were centrifuged at 100,000 g for 30 minutes to separate the membrane fraction (pellet) and the cytosolic fraction (supernatant). NOX activity was measured by lucigeninderived chemiluminescence, with 100 μmol/L NADPH or NADH as substrate, 50 μmol/L lucigenin, and 25 μg of cell membrane proteins. Chemiluminescence was measured using a luminometer (Turner Designs, Sunnyvale, CA, USA) for 1 minute. The signal was normalized and expressed as arbitrary light units per microgram protein per minute. Rac activity
The Rac activity assay was performed using the Rac-GEF (guanine-nucleotide exchange factors) Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, cells were washed in cold PBS, lysed in 1× Assay/Lysis Buffer, and centrifuged
Wang et al. Chinese Journal of Cancer (2015) 34:8
Page 3 of 11
for 10 minutes at 14,000 g at 4°C. Aliquots from the supernatant were used for determining protein concentration. The supernatant was incubated with nucleotide-free Rac1 G15A agarose beads to pull down the active form of RacGEFs. The beads were washed 3 times with 1× Assay/Lysis Buffer, and the bound proteins were eluted. The active Rac proteins were detected by Western blotting using an antiRac-GEF antibody (Tiam1).
of 0.9% sodium chloride solution by intraperitoneal injection. Five weeks after inoculation, all mice were euthanized and the tumor weights were measured. Animal experiments were approved by Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center and performed under the guidelines of the Care and Use of Laboratory Animals (NIH publications Nos. 80–23, revised 1996).
Invasion assay
Immunohistochemistry and TUNEL assay
Invasion assays were performed with BD BioCoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA). Pre-coated filter Matrigel inserts were rehydrated with 0.5 mL of PBS for 2 hours in humidified tissue culture incubator at 37°C in 5% CO2 atmosphere. After rehydration, PBS was removed. Then, 1 × 105 parental or K-ras–transformed E6E7 cells and Capan-1, AsPC-1, Panc-1 cells in 0.5 mL of supplement-free medium with or without 10 μmol/L capsaicin were seeded onto the upper part of each chamber insert, and the 24-well plates were filled with 0.5 mL of their culture medium. Following incubation for 16 hours, noninvaded cells on the upper surface of the insert were wiped off with a cotton swab, and the cells that had migrated onto the lower surface of the filter, were fixed and stained with the Hema 3 Manual Staining System (Fisher Scientific, Pittsburgh, PA, USA) containing a fixative and 2 stain solutions. The inserts were air dried and photographed. Invasiveness was determined by counting cells in 3 microscopic fields (×100) per well, and the extent of invasion was expressed as an average number of cells per microscopic field.
Representative tumor tissues were sectioned and embedded in paraffin. The slides were then incubated with the primary antibody (mouse anti–8-oxoguanine monoclonal antibody, Abcam, Cambridge, UK) at 1:200 dilution overnight in a humidified chamber at 4°C. The slides were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Envision Detection Kit, Dako, Glostrup, Denmark) at 37°C for 30 minutes. Finally, the samples were stained with 3, 3-diaminobenzidine (DAB) solution and counterstained with hematoxylin and eosin (HE). Tumor cell death induced by capsaicin was detected by TUNEL assay with the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN, USA) according to manufacturer’s instructions. Statistical analysis
Statistical significant differences were evaluated by using Student’s t test (Prism GraphPad, San Diego, CA, USA). The Kolmogorov-Smirnov test (Cell Quest Pro software, Becton-Dickinson, San Jose, CA, USA) was used to evaluate the significant difference between control and treatment groups in flow cytometry analysis. A P value of
